PROJECT SUMMARY Chronic HCV infection afflicts 180 million individuals throughout the world. The majority of individuals infected with HCV develop persistence, suggesting that HCV successfully subverts innate and adaptive immune responses. Mathematical models have projected a massive increase in the number of patients developing HCV-related complications (e.g., cirrhosis, hepatocellular carcinoma) in the next few decades as the population ages. Although hepatocytes comprise the majority of the total cell population within the liver, the non- parenchymal liver cells have been increasingly recognized as playing central roles in chronic inflammation and complications from HCV infection. Although the development of direct-acting antivirals (DAAs) has led to nearly miraculous cure rates [compared to interferon (IFN)-based therapy], a number of important questions remain unanswered that demand further study. To what extent is immunity restored in patients cured with DAAs and what are the effects on cross-talk between the multiple cell types within the hepatic microenvironment? What effect does HCV cure have on inflammation, fibrosis development, and further hepatic decompensation? The answers to these questions have remained elusive and have implications for other liver disease. Here, we will define the breadth and pace of reconstitution of hepatic and peripheral innate and adaptive immunity induced by DAA therapy in patients with chronic HCV infection. We will also determine how DAA-mediated viral eradication affects epigenetic reprogramming of HCV-specific CTLs. A large cohort of already-enrolling patients, including those who have previously failed DAA therapy and have evidence of resistance-associated variants, will be serially studied according to pre-treatment viral level and fibrosis stage. Next, we will characterize the complex communication between hepatocytes and nonparenchymal cells (NPCs) in HCV infection and the impact of DAA-mediated viral elimination. We hypothesize that exosomes play important roles in intercellular communication that is intricately linked to the cell origin of the exosomes, as well as the biologic milieu; exosomes can shuttle HCV and biologically active molecules from infected hepatoma cells to a variety of NPC. We will examine how purified exosomes derived from HCV-infected and DAA-cured hepatocytes differentially trigger responses in Kupffer cells (KCs), liver sinusoidal endothelial cells (LSECs) and HSCs. We will comprehensively characterize the exosomal content following HCV exposure and determine which components, including microRNAs, either amplify or attenuate inflammatory or fibrotic pathways. If successful, these studies would provide novel insights into the mechanisms mediating viral clearance, residual inflammation, and liver injury (fibrosis) and have broad-reaching relevance in the rapidly changing field of HCV, potentially opening up new avenues of research into this highly prevalent liver disease worldwide.